Microbiology Lab Concepts and Core Topics: Metabolism, Genetics, and Biotechnology

Study Guide - Smart Notes

Tailored notes based on your materials, expanded with key definitions, examples, and context.

Lab Concepts

Streak Plating

Streak plating is a fundamental microbiological technique used to isolate pure colonies of microorganisms from a mixed sample.

Purpose: To obtain isolated colonies for further study or identification.
Common Errors: Not flaming the loop between streaks, using too much inoculum, or overlapping streaks can prevent isolation.
Troubleshooting: Ensure proper sterilization of the loop, use gentle pressure, and streak in quadrants to dilute the sample effectively.

DNA Extraction

DNA extraction is the process of isolating DNA from cells for analysis. It involves breaking open cells, removing proteins and other contaminants, and precipitating DNA.

Centrifuge Safety and Balancing: Always balance tubes with equal volumes opposite each other to prevent damage or accidents.
Centrifuge Purpose: To separate components based on density; heavier particles form a pellet, while lighter materials remain in the supernatant.
Supernatant vs Pellet: The supernatant is the liquid above the pellet after centrifugation; the pellet is the solid material at the bottom.

Key Materials and Functions in Strawberry DNA Extraction

Strawberries: High DNA content due to being octoploid.
Dish Soap (Detergent): Dissolves cell and nuclear membranes, releasing DNA.
Salt (NaCl): Neutralizes DNA charge, helps DNA clump, and separates proteins.
Ice-Cold Ethanol/Isopropyl Alcohol: Precipitates DNA, making it visible.
Plastic Bag/Mash: Mechanically breaks cell walls.
Filter: Removes debris, allowing DNA to pass through in the filtrate.
Stirrer: Collects precipitated DNA.

Comparison of DNA Extraction Methods

Bead Bashing: Physically disrupts cells using beads.
DNA Wash: Removes contaminants from DNA.
Column Filtration: DNA binds to a column, impurities are washed away, and DNA is eluted.

Additional info: These methods are chosen based on sample type and downstream applications.

CH 5: Microbial Metabolism

Catabolism and Anabolism

Microbial metabolism consists of all chemical reactions within a microorganism, divided into catabolism and anabolism.

Catabolism: Breakdown of complex molecules into simpler ones, releasing energy (exergonic, hydrolytic reactions).
Anabolism: Synthesis of complex molecules from simpler ones, consuming energy (endergonic, dehydration synthesis).

ATP in Metabolic Reactions

ATP Release: Catabolic reactions release ATP.
ATP Consumption: Anabolic reactions consume ATP.

Enzymes

Enzymes are biological catalysts, usually proteins ending in "-ase," that speed up chemical reactions without being consumed.

Role in Pathways: Enzymes lower activation energy and are essential for metabolic pathways.
Substrate: The molecule upon which an enzyme acts.
Active Site: The region on the enzyme where the substrate binds.
Products: Common products include ATP, acids, gases, and alcohols.

Factors Affecting Enzyme Activity

pH: Extreme pH can denature enzymes.
Temperature: High temperatures can denature enzymes; low temperatures slow reactions.
Substrate Concentration: Higher concentrations increase reaction rate up to a saturation point.
Inhibitors: Competitive and noncompetitive inhibitors can decrease enzyme activity.

CH 8: Microbial Genetics

Central Dogma and -Omics Analyses

The central dogma describes the flow of genetic information: DNA → RNA → Protein.

Genomics: Study of the entire genome (DNA).
Transcriptomics: Study of RNA transcripts.
Proteomics: Study of the protein complement.
Mutations: Changes at any step can affect subsequent steps and cellular function.

Gene Expression and Operons

Transcription: Synthesis of RNA from DNA; gene expression.
Constitutively Expressed: Genes that are always active.
Repressible Operon: Normally on; can be turned off by a repressor (e.g., trp operon).
Inducible Operon: Normally off; can be turned on by an inducer (e.g., lac operon).
Enzymes and Substrates: Induction and repression depend on the presence or absence of substrates or end products.
Operon: A cluster of genes under control of a single promoter.
Operator: DNA segment where a repressor binds.
Promoter: DNA sequence where RNA polymerase binds to initiate transcription.

Enzymes in Genetic Processes

Enzymes are essential for DNA replication, transcription, and translation.

RNA Differences: Prokaryotes vs Eukaryotes

Prokaryotic RNA: Often polycistronic (multiple genes per mRNA), no introns, transcription and translation are coupled.
Eukaryotic RNA: Monocistronic, contains introns (spliced out), transcription in nucleus, translation in cytoplasm.

Ribosomes and Amino Acids

Ribosomes: Sites of protein synthesis; differ in size between prokaryotes (70S) and eukaryotes (80S).
Amino Acids: Building blocks of proteins; sequence determined by mRNA codons.

Codons

Definition: A codon is a sequence of three nucleotides in mRNA that codes for a specific amino acid.
Universality: Most codons are universal, but some variations exist in mitochondria and some microbes.

Gene Transfer and Mobile Genetic Elements

Vertical Gene Transfer: Genes passed from parent to offspring.
Horizontal Gene Transfer: Genes transferred between organisms in the same generation.
Mobile Genetic Elements (MGEs): DNA segments that can move within or between genomes (e.g., plasmids, transposons).

Plasmids

Conjugative Plasmids: Carry genes for transfer between cells (e.g., F factor).
R Factors: Plasmids carrying antibiotic resistance genes.

Transposons

Function: DNA sequences that can change position within the genome.
Pros: Increase genetic diversity.
Cons: Can disrupt gene function, cause mutations.

Phages

Lytic Cycle: Phage replicates and lyses host cell.
Lysogenic Cycle: Phage DNA integrates into host genome and replicates with it.

Mechanisms of Horizontal Gene Transfer

Conjugation: Direct transfer of DNA via cell-to-cell contact.
Transformation: Uptake of free DNA from the environment (e.g., Griffith's experiment).
Transduction: Transfer of DNA by bacteriophages.

CH 9: Biotechnology and DNA Technology

Key Concepts

Vector: DNA molecule used to carry foreign genetic material into a host cell (e.g., plasmids, viruses).
Biotechnology: Use of living organisms or their products for practical purposes.
rDNA Technology: Techniques for combining DNA from different sources (genetic modification).
Common Products: Insulin, growth hormones, vaccines, genetically modified crops.

Recombination

Definition: Exchange of genetic material between different DNA molecules.
Result: New genetic combinations, increased diversity.

PCR (Polymerase Chain Reaction)

Purpose: Amplifies specific DNA sequences.
Heating: Denatures DNA strands.
Cooling: Allows primers to anneal and DNA polymerase to extend new strands.

Cloning

Purpose: Producing identical copies of DNA, cells, or organisms for research, medicine, or agriculture.

DNA Insertion Methods

Transformation: Uptake of naked DNA by cells.
Electroporation: Electric pulses create pores in cell membranes for DNA entry.
Protoplast Fusion: Fusion of cells without cell walls to combine genetic material.
Microinjection: Direct injection of DNA into cells using a fine needle.

Sequencing Methods

Amplicon Sequencing: Targets specific DNA regions for sequencing (e.g., 16S rRNA gene).
Shotgun Sequencing: Randomly sequences all DNA in a sample; useful for whole-genome or metagenomic studies.
Nanotechnology: Used to enhance sequencing accuracy and throughput.

Method	Main Purpose	Key Features
Amplicon Sequencing	Targeted sequencing of specific genes	High specificity, lower cost, limited to known regions
Shotgun Sequencing	Whole-genome/metagenome sequencing	Comprehensive, detects unknowns, higher cost

Additional info: Modern sequencing platforms (e.g., nanopore, Illumina) are used for both approaches.